Fig. 4.

Label free imaging. (a) Energy diagrams and wavelengths in two-photon fluorescence (2PM), second harmonic generation (SHG), and third harmonic generation (THG) imaging. (b) THG image of striatum in a mouse brain (coronal section), showing white-matter fibers and neurons. The bright grainy structures are axon bundles that run perpendicular to the image plane. (c) Merged THG (green) and SHG (red) signal of a mouse corpus callosum. White-matter structures are visible in both SHG and THG images, but gray matter is only visible in the THG image. THG signals originate mostly from the myelin sheaths surrounding axons, whereas SHG signals are produced by polarized microtubules inside the axons. (d) Sagittal THG image of a zebrafish embryo during the 512 cell stage. The THG signal, generated mostly from the lipids in the plasma membrane, highlights cell contours and reveals the yolk-blastoderm interface as indicated by the white arrowheads. (e) Temporal sequence (0, 2 and 8 minutes) of βactin:H2B/mcherry transgenic zebrafish embryo, highlighting mitosis, simultaneously with SHG (green), 2PM (red) and THG (blue). (f) Gastrulating Drosophila embryos with contrast derived from THG (white) and 2PM (mRFP-red, EGFP-green, and autofluorescence-blue) imaging. Panels b-c are adapted from S. Witte, et al. [50] with permission from PNAS; panel d-e are adapted from N. Olivier, et al. [51] with permission from Science; and panel f is adapted from P. Mahou, et al. [52] with permission from Nature.